*Target (protein/gene name): Histidinol Phosphatase (Histidine biosynthesis bifunctional protein hisB) *NCBI Gene # or RefSeq#: GI:114793851 *Protein ID (NP or XP #) or Wolbachia#: NP_288527.2 *Organism (including strain):Escherichia coli O157:H7 Etiologic Risk Group (see link below):Risk Group 1 *Background/Disease Information (sort of like the Intro to your Mini Research Write up):
The bacterium Escherichia coli O157:H7 is a worldwide threat to public health and has been implicated in many outbreaks of haemorrhagic colitis. In addition to causing diarrhea, Escherichia coli O157:H7 infection can lead to hemolytic-uremic syndrome (HUS), a severe disease characterized by hemolysis and renal failure. The severity of disease, the lack of effective treatment and the potential for large-scale outbreaks from contaminated food supplies have propelled intensive research on the pathogenesis and detection of E. coli O157:H7. Essentiality of this protein: Histidinol Phosphatase is not essential. Complex of proteins?: No Druggable Target: There are no current druggable targets.
1,387 new genes encoded in strain-specific clusters of diverse sizes were found in O157:H7. These include candidate virulence factors, alternative metabolic capacities, several prophages and other new functions--all of which could be targets for surveillance. Genome Sequence Article *EC#: 3.1.3.15 Link to BRENDA EC# page:http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.3.15&Suchword=&organism%5B%5D=Escherichia+coli&show_tm=0 Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): Spectrophotometric
High-resolution phylogenomic approaches allow the dynamics of pathogenome evolution to be followed at a high level of phylogenetic accuracy and resolution. SNP discovery and study of genome architecture and prophage content identified numerous biomarkers to assess the extent of genetic diversity within a set of clinical and environmental strains. Genomic Anatomy Article -- link to Sigma (or other company) page for assay or assay reagents (substrates)
This information is not yet available and could not be found -- link (or citation) to paper that contains assay information (Page 4598)http://iai.asm.org/content/75/9/4597.full.pdf -- List cost and quantity of substrate reagents and supplier
LB Media (Glucose)-----$40.70 L3022-250G
Indole-------------------------$64.30 I3408-100G
Sigma Aldrich Supplier Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 2FPW
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB 2FPU vs. 2FPW
---- Query Coverage: 176 letters
---- Max % Identities: 163/163 (100%)
---- % Positives: 163/163 (100%)
---- Chain used for homology: B
Current Inhibitors: HisB-N is active in the presence of Mg(2+), Mn(2+), Co(2+), or Zn(2+), but Ca(2+) has an inhibitory effect.Article Source Expression Information (has it been expressed in bacterial cells): Yes, it has been expressed in E. Coli BL21 (DE3). Article Source Purification Method: Purification Method is explained in the Cloning, Mutagenesis, and Purification section of the article below. http://www.jbc.org/content/281/49/37930.full.pdf Image of protein (PyMol with features delineated and shown separately):
Crystal Structure of the N-terminal domain of E.coli HisB- Complex with histidinol
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): MGSSHHHHHHGSSQKYLFIDRDGTLISEPPSDFQVDRFDKLAFEPGVIPQLLKLQKAGYKLVMITNQDGL GTQSFPQADFDGPHNLMMQIFTSQGVQFDEVLICPHLPADECDCRKPKVKLVERYLAEQAMDRANSYVIG DRATDIQLAENMGINGLRYDRETLNWPMIGEQLTRR *length of your protein in Amino Acids: 176 Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 20040.7 Molar Extinction coefficient of your protein at 280 nm wavelength: 13075 http://web.expasy.org/cgi-bin/protparam/protparam
Abs 0.1% (=1 g/l) 0.652, assuming all pairs of Cys residues form cystines TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
TMpred Graph Image of Amino Acid Sequence of Histidinol Phosphatase
NCBI Gene *CDS Gene Sequence (paste as text only): >gi|16445223:2835746-2836813 Escherichia coli O157:H7 str. EDL933 chromosome, complete genome ATGAGTCAGAAGTATCTTTTTATCGATCGCGATGGAACCCTGATTAGCGAACCGCCGAGTGATTTTCAGG TGGATCGTTTTGACAAACTCGCCTTTGAACCGGGCGTGATCCCGCAACTGCTGAAGCTGCAAAAAGCGGG CTACAAACTGGTGATGATCACTAATCAGGATGGTCTGGGAACACAAAGTTTCCCGCAGGCGGATTTCGAT GGCCCGCACAACCTGATGATGCAGATATTCACYTCGCAAGGCGTACAGTTTGATGAAGTGCTGATTTGTC CGCACCTGCCCGCCGATGAGTGCGACTGCCGTAAACCGAAAGTAAAACTGGTGGAGCGTTATCTCGCTGA GCAAGCGATGGATCGCGCCAACAGTTATGTGATTGGCGATCGCGCGACCGATATTCAACTCGCTGAAAAC ATGGGCATTAATGGTTTACGCTACGACCGCGAAACCCTGAACTGGCCAATGATTGGCGAGCAACTCACCA GACGTGACCGTTACGCTCACGTAGTGCGTAATACCAAAGAGACGCAGATTGACGTTCAGGTGTGGCTGGA TCGTGAAGGTGGCAGCAAGATTAATACCGGCGTTGGCTTCTTTGATCACATGCTGGATCAGATCGCCACC CACGGCGGTTTCCGTATGGAAATCAACGTCAAAGGCGACCTCTATATCGACGATCACCACACCGTCGAAG ATACCGGCCTGGCGCTGGGCGAAGCGTTAAAAATTGCCCTCGGCGATAAACGCGGTATTTGCCGCTTTGG TTTTGTGCTGCCGATGGACGAATGCCTTGCCCGCTGCGCGCTGGATATCTCTGGTCGCCCGCACCTGGAA TATAAAGCCGAGTTTACCTACCAACGCGTGGGCGATCTCAGCACCGAGATGATCGAGCACTTCTTCCGTT CGCTCTCTTACACCATGGGCGTGACGCTCCACCTGAAAACCAAAGGTAAAAACGATCATCACCGTGTAGA GAGCCTGTTCAAAGCCTTTGGTCGGACCCTGCGCCAGGCCATCCGCGTGGAAGGCGATACCCTGCCCTCG TCGAAAGGAGTGCTGTAA *GC% Content for gene: 53.18%
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
Target-Histidinol Phosphatase
*Target (protein/gene name): Histidinol Phosphatase (Histidine biosynthesis bifunctional protein hisB)*NCBI Gene # or RefSeq#: GI:114793851
*Protein ID (NP or XP #) or Wolbachia#: NP_288527.2
*Organism (including strain): Escherichia coli O157:H7
Etiologic Risk Group (see link below): Risk Group 1
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
The bacterium Escherichia coli O157:H7 is a worldwide threat to public health and has been implicated in many outbreaks of haemorrhagic colitis. In addition to causing diarrhea, Escherichia coli O157:H7 infection can lead to hemolytic-uremic syndrome (HUS), a severe disease characterized by hemolysis and renal failure. The severity of disease, the lack of effective treatment and the potential for large-scale outbreaks from contaminated food supplies have propelled intensive research on the pathogenesis and detection of E. coli O157:H7.
Essentiality of this protein: Histidinol Phosphatase is not essential.
Complex of proteins?: No
Druggable Target: There are no current druggable targets.
1,387 new genes encoded in strain-specific clusters of diverse sizes were found in O157:H7. These include candidate virulence factors, alternative metabolic capacities, several prophages and other new functions--all of which could be targets for surveillance. Genome Sequence Article
*EC#: 3.1.3.15
Link to BRENDA EC# page: http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.3.15&Suchword=&organism%5B%5D=Escherichia+coli&show_tm=0
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): Spectrophotometric
High-resolution phylogenomic approaches allow the dynamics of pathogenome evolution to be followed at a high level of phylogenetic accuracy and resolution. SNP discovery and study of genome architecture and prophage content identified numerous biomarkers to assess the extent of genetic diversity within a set of clinical and environmental strains. Genomic Anatomy Article
-- link to Sigma (or other company) page for assay or assay reagents (substrates)
This information is not yet available and could not be found
-- link (or citation) to paper that contains assay information
(Page 4598) http://iai.asm.org/content/75/9/4597.full.pdf
-- List cost and quantity of substrate reagents and supplier
LB Media (Glucose)-----$40.70 L3022-250G
Indole-------------------------$64.30 I3408-100G
Sigma Aldrich Supplier
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 2FPW
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
2FPU vs. 2FPW
---- Query Coverage: 176 letters
---- Max % Identities: 163/163 (100%)
---- % Positives: 163/163 (100%)
---- Chain used for homology: B
Current Inhibitors: HisB-N is active in the presence of Mg(2+), Mn(2+), Co(2+), or Zn(2+), but Ca(2+) has an inhibitory effect.Article Source
Expression Information (has it been expressed in bacterial cells): Yes, it has been expressed in E. Coli BL21 (DE3). Article Source
Purification Method: Purification Method is explained in the Cloning, Mutagenesis, and Purification section of the article below.
http://www.jbc.org/content/281/49/37930.full.pdf
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MGSSHHHHHHGSSQKYLFIDRDGTLISEPPSDFQVDRFDKLAFEPGVIPQLLKLQKAGYKLVMITNQDGL GTQSFPQADFDGPHNLMMQIFTSQGVQFDEVLICPHLPADECDCRKPKVKLVERYLAEQAMDRANSYVIG DRATDIQLAENMGINGLRYDRETLNWPMIGEQLTRR
*length of your protein in Amino Acids: 176
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 20040.7
Molar Extinction coefficient of your protein at 280 nm wavelength: 13075
http://web.expasy.org/cgi-bin/protparam/protparam
Abs 0.1% (=1 g/l) 0.652, assuming all pairs of Cys residues form cystines
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
NCBI Gene
*CDS Gene Sequence (paste as text only):
>gi|16445223:2835746-2836813 Escherichia coli O157:H7 str. EDL933 chromosome, complete genome
ATGAGTCAGAAGTATCTTTTTATCGATCGCGATGGAACCCTGATTAGCGAACCGCCGAGTGATTTTCAGG
TGGATCGTTTTGACAAACTCGCCTTTGAACCGGGCGTGATCCCGCAACTGCTGAAGCTGCAAAAAGCGGG
CTACAAACTGGTGATGATCACTAATCAGGATGGTCTGGGAACACAAAGTTTCCCGCAGGCGGATTTCGAT
GGCCCGCACAACCTGATGATGCAGATATTCACYTCGCAAGGCGTACAGTTTGATGAAGTGCTGATTTGTC
CGCACCTGCCCGCCGATGAGTGCGACTGCCGTAAACCGAAAGTAAAACTGGTGGAGCGTTATCTCGCTGA
GCAAGCGATGGATCGCGCCAACAGTTATGTGATTGGCGATCGCGCGACCGATATTCAACTCGCTGAAAAC
ATGGGCATTAATGGTTTACGCTACGACCGCGAAACCCTGAACTGGCCAATGATTGGCGAGCAACTCACCA
GACGTGACCGTTACGCTCACGTAGTGCGTAATACCAAAGAGACGCAGATTGACGTTCAGGTGTGGCTGGA
TCGTGAAGGTGGCAGCAAGATTAATACCGGCGTTGGCTTCTTTGATCACATGCTGGATCAGATCGCCACC
CACGGCGGTTTCCGTATGGAAATCAACGTCAAAGGCGACCTCTATATCGACGATCACCACACCGTCGAAG
ATACCGGCCTGGCGCTGGGCGAAGCGTTAAAAATTGCCCTCGGCGATAAACGCGGTATTTGCCGCTTTGG
TTTTGTGCTGCCGATGGACGAATGCCTTGCCCGCTGCGCGCTGGATATCTCTGGTCGCCCGCACCTGGAA
TATAAAGCCGAGTTTACCTACCAACGCGTGGGCGATCTCAGCACCGAGATGATCGAGCACTTCTTCCGTT
CGCTCTCTTACACCATGGGCGTGACGCTCCACCTGAAAACCAAAGGTAAAAACGATCATCACCGTGTAGA
GAGCCTGTTCAAAGCCTTTGGTCGGACCCTGCGCCAGGCCATCCGCGTGGAAGGCGATACCCTGCCCTCG
TCGAAAGGAGTGCTGTAA
*GC% Content for gene: 53.18%
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
References:
http://www.ncbi.nlm.nih.gov/pubmed/19564389/